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1.  Genetic Diversity of Japanese Encephalitis Virus Isolates Obtained from the Indonesian Archipelago Between 1974 and 1987 
Five genotypes (GI–V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI–III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate of the Indonesia/Malaysia region, together with its plethora of distinct fauna and flora, may have driven the emergence and evolution of JEV. This is consistent with the extensive genetic diversity seen among the JEV isolates observed in this study, and further substantiates the hypothesis that JEV originated in the Indonesia-Malaysia region.
PMCID: PMC3700436  PMID: 23590316
Indonesia; Japanese encephalitis virus; Molecular epidemiology
2.  Tick-Borne Pathogens in Ticks Feeding on Migratory Passerines in Western Part of Estonia 
During southward migration in the years 2006–2009, 178 migratory passerines of 24 bird species infested with ticks were captured at bird stations in Western Estonia. In total, 249 nymphal ticks were removed and analyzed individually for the presence of Borrelia burgdorferi sensu lato (s.l.), tick-borne encephalitis virus (TBEV), and Anaplasma phagocytophilum. The majority of ticks were collected from Acrocephalus (58%), Turdus (13%), Sylvia (8%), and Parus (6%) bird species. Tick-borne pathogens were detected in nymphs removed from Acrocephalus, Turdus, and Parus bird species. TBEV of the European subtype was detected in 1 I. ricinus nymph removed from A. palustris. B. burgdorferi s.l. DNA was found in 11 ticks (4.4%) collected from Turdus and Parus species. Bird-associated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodent-associated B. afzelii was detected in 3 I. ricinus nymphs from 2 P. major birds. One of the B. afzelii-positive nymphs was infected with a mix of 2 B. afzelii strains, whereas 1 of these strains was also detected in another nymph feeding on the same great tit. The sharing of the same B. afzelii strain by 2 nymphs indicates a possible transmission of B. afzelii by co-feeding on a bird. A. phagocytophilum DNA was detected in 1 I. ricinus nymph feeding on a T. iliacus. The results of the study confirm the possible role of migratory birds in the dispersal of ticks infected with tick-borne pathogens along the southward migration route via Estonia.
PMCID: PMC3700471  PMID: 23590318
Tick-borne pathogen; TBEV; Borrelia; Reservoir; tick; Ixodidae; Bird
3.  Detection of Rickettsia parkeri from within Piura, Peru, and the First Reported Presence of Candidatus Rickettsia andeanae in the Tick Rhipicephalus sanguineus 
Domestic farm animals (n=145) were sampled for the presence of ectoparasites in northwestern Peru during March, 2008. Ninety domestic animals (62%) were positive for the presence of an ectoparasite(s) and produced a total collection of the following: 728 ticks [Amblyomma maculatum, Anocentor nitens, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus, and Otobius megnini], 12 lice (Haematopinus suis), and 3 fleas (Ctenocephalides felis). A Rickettsia genus-specific qPCR assay was performed on nucleic acid preparations of the collected ectoparasites that resulted in 5% (37/743, 35 ticks and 2 fleas) of the ectoparasites positive for the presence of Rickettsia. DNA from the positive individual ticks was tested with 2 other qPCR assays for the presence of the ompB gene in Candidatus Rickettsia andeanae or Rickettsia parkeri. Candidatus R. andeanae was found in 25 A. maculatum ticks and in two Rh. sanguineus ticks, whereas R. parkeri was detected in 6 A. maculatum ticks. Two A. maculatum were co-infected with both Candidatus R. andeanae and R. parkeri. Rickettsia felis was detected in 2 fleas, Ctenocephalides felis, by multilocus sequence typing of the 17-kD antigen and ompA genes. These findings expand the geographic range of R. parkeri to include Peru as well as expand the natural arthropod vector of Candidatus R. andeanae to include Rhipicephalus sanguineus.
PMCID: PMC3700472  PMID: 23488453
Candidatus Rickettsia andeanae; Rickettsia parkeri; Ticks; Peru
4.  Estimating West Nile Virus Transmission Period in Pennsylvania Using an Optimized Degree-Day Model 
We provide calibrated degree-day models to predict potential West Nile virus (WNV) transmission periods in Pennsylvania. We begin by following the standard approach of treating the degree-days necessary for the virus to complete the extrinsic incubation period (EIP), and mosquito longevity as constants. This approach failed to adequately explain virus transmission periods based on mosquito surveillance data from 4 locations (Harrisburg, Philadelphia, Pittsburgh, and Williamsport) in Pennsylvania from 2002 to 2008. Allowing the EIP and adult longevity to vary across time and space improved model fit substantially. The calibrated models increase the ability to successfully predict the WNV transmission period in Pennsylvania to 70–80% compared to less than 30% in the uncalibrated model. Model validation showed the optimized models to be robust in 3 of the locations, although still showing errors for Philadelphia. These models and methods could provide useful tools to predict WNV transmission period from surveillance datasets, assess potential WNV risk, and make informed mosquito surveillance strategies.
PMCID: PMC3700474  PMID: 23590317
West Nile virus; Phenology model; Degree-days; Extrinsic incubation period; Mosquito longevity
5.  Serological Evidence of Toxoplasma gondii Infection in Five Species of Bats in China 
Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect almost all warm-blooded animals and humans with a worldwide distribution. Bats are reservoirs for an increasing number of emerging zoonotic viruses, such as henipaviruses and severe acute respiratory syndrome coronavirus (SARS-CoV). However, little is known of T. gondii infection in bats. The objective of the present study was to determine the seroprevalence of T. gondii infection in bats in China. A total of 217 serum samples from 5 species of bats were collected between April, 2010, and August, 2011, from 4 provinces in China. Antibodies to T. gondii were determined using the modified agglutination test (MAT, 1:25 or higher). Antibodies to T. gondii were found in 26.5% (18/68) Megaderma lyra, 13.6% (12/88) Rousettus leschenaulti, 13.6% (3/22) Cynopterus sphinx, 20% (4/20) Vespertilio superaus, and 15.8% (3/19) Pipistrellus javanicus. Antibody titers ranged from 1:25 to 1:400, with titers of 1:200 detected in 4 of the 5 bat species. The present study suggests the likely occurrence of T. gondii infection in bats in China, and these bats are new putative hosts for T. gondii, which may pose a threat to human health.
PMCID: PMC3669597  PMID: 23473226
Toxoplasma gondii; Toxoplasmosis; Bat; China; Seroprevalence; Modified agglutination test
6.  Chronic Infections of West Nile Virus Detected in California Dead Birds 
During 2010 and 2011, 933 recently deceased birds, submitted as part of the dead bird surveillance program, tested positive for West Nile virus RNA at necropsy. The relative amount of RNA measured by qRT-PCR cycles ranged from 8.2 to 37.0 cycle threshold (Ct) and formed a bimodal frequency distribution, with maxima at 20 and 36 Ct and minima at 28–30 Ct. On the basis of frequency distributions among different avian species with different responses to infection following experimental inoculation, field serological data indicating survival of infection, and the discovery of persistent RNA in experimentally infected birds, dead birds collected in nature were scored as “recent” or “chronic” infections on the basis of Ct scores. The percentage of birds scored as having chronic infections was highest during late winter/spring, when all birds were after hatching year, and lowest during late summer, when enzootic transmission was typically highest as indicated by mosquito infections. Our data indicated that intervention efforts should not be based on dead birds with chronic infections unless supported by additional surveillance metrics.
PMCID: PMC3669600  PMID: 23488452
Surveillance; West Nile virus; Dead birds; Chronic infections; Overwintering
7.  Genetic and Biological Characterization of Tick-Borne Encephalitis Virus Isolated from Wild Rodents in Southern Hokkaido, Japan in 2008 
Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A recent epizootiological survey indicated that endemic foci of TBEV have been maintained in the southern part of Hokkaido until recently. In this study, we sought to isolate TBEV from wild rodents in the area. One virus, designated Oshima 08-As, was isolated from an Apodemus speciosus captured in Hokuto in 2008. Oshima 08-As was classified as the Far Eastern subtype of TBEV and formed a cluster with the other strains isolated in Hokkaido from 1995 to 1996. Thirty-six nucleotide differences resulted in 12 amino acid changes between Oshima 08-As and Oshima 5–10 isolated in 1995. Oshima 08-As caused high mortality and morbidity in a mouse model compared with Oshima 5–10. Although similar transient viral multiplication in the spleen was observed in the mice infected with Oshima 08-As and Oshima 5–10, greater viral multiplication with an inflammatory response was noted in the brains of mice infected with Oshima 08-As than those infected with Oshima 5–10. These data indicate that a few naturally occurring mutations affect the pathogenicity of the Oshima strains endemic in the southern part of Hokkaido.
PMCID: PMC3669602  PMID: 23590320
Tick-borne encephalitis virus; Oshima 08-As; Oshima 5–10; Hokkaido
8.  Serologic Evidence for Borrelia hermsii Infection in Rodents on Federally Owned Recreational Areas in California 
Tick-borne relapsing fever (TBRF) is endemic in mountainous regions of the western United States. In California, the principal agent is the spirochete Borrelia hermsii, which is transmitted by the argasid tick Ornithodoros hermsi. Humans are at risk of TBRF when infected ticks leave an abandoned rodent nest in quest of a blood meal. Rodents are the primary vertebrate hosts for B. hermsii. Sciurid rodents were collected from 23 sites in California between August, 2006, and September, 2008, and tested for serum antibodies to B. hermsii by immunoblot using a whole-cell sonicate and a specific antigen, glycerophosphodiester phosphodiesterase (GlpQ). Antibodies were detected in 20% of rodents; seroprevalence was highest (36%) in chipmunks (Tamias spp). Seroprevalence in chipmunks was highest in the Sierra Nevada (41%) and Mono (43%) ecoregions and between 1900 and 2300 meters elevation (43%). The serological studies described here are effective in implicating the primary vertebrate hosts involved in the maintenance of the ticks and spirochetes in regions endemic for TBRF.
PMCID: PMC3669604  PMID: 23488454
Borrelia hermsii; Tick-borne relapsing fever; Rodentia
9.  The Novel H7N9 Influenza A Virus: Its Present Impact and Indeterminate Future 
PMCID: PMC3709519  PMID: 23631726
10.  Presence, Distribution, and Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Small Animal Teaching Hospital: A Year-Long Active Surveillance Targeting Dogs and Their Environment 
Methicillin-resistant Staphylococcus aureus (MRSA) is known to be present in small animal veterinary clinical environments. However, a better understanding of the ecology and dynamics of MRSA in these environments is necessary for the development of effective infectious disease prevention and control programs. To achieve this goal, a yearlong active MRSA surveillance program was established at The Ohio State University (OSU) Veterinary Medical Center to describe the spatial and molecular epidemiology of this bacterium in the small animal hospital. Antimicrobial susceptibility testing, staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE) typing, and dendrogram analysis were used to characterize and analyze the 81 environmental and 37 canine-origin MRSA isolates obtained during monthly sampling events. Overall, 13.5% of surfaces were contaminated with MRSA at 1 or more sampling times throughout the year. The majority of the environmental and canine isolates were SCCmec type II (93.8% and 86.5%, respectively) and USA100 (90.1% and 86.5%, respectively). By PFGE analysis, these isolates were found to be closely related, which reflects a low diversity of MRSA strains circulating in the hospital. For 5 consecutive months, 1 unique pulsotype was the most prevalent across the medical services and was recovered from a variety of surfaces and hospital locations. Carts/gurneys, doors, and examination tables/floors were the most frequently contaminated surfaces. Some surfaces maintained the same pulsotypes for 3 consecutive months. Molecular analysis found that incoming MRSA-positive dogs were capable of introducing a new pulsotype into the hospital environment during the surveillance period. Our results suggest that once a MRSA strain is introduced into the hospital environment, it can be maintained and spread for extended periods of time. These findings can aid in the development of biosecurity and biocontainment protocols aimed at reducing environmental contamination and potential exposures to MRSA in veterinary hospital staff, clients, and patients.
PMCID: PMC3636584  PMID: 23473216
MRSA; Surveillance; Molecular epidemiology; Veterinary hospitals; Environment
11.  Co-Infection and Genetic Diversity of Tick-Borne Pathogens in Roe Deer from Poland 
Wild species are essential hosts for maintaining Ixodes ticks and the tick-borne diseases. The aim of our study was to estimate the prevalence, the rate of co-infection with Babesia, Bartonella, and Anaplasma phagocytophilum, and the molecular diversity of tick-borne pathogens in roe deer in Poland. Almost half of the tested samples provided evidence of infection with at least 1 species. A. phagocytophilum (37.3%) was the most common and Bartonella (13.4%) the rarest infection. A total of 18.3% of all positive samples from roe deer were infected with at least 2 pathogens, and one-third of those were co-infected with A. phagocytophilum, Bartonella, and Babesia species. On the basis of multilocus molecular studies we conclude that: (1) Two different genetic variants of A. phagocytophilum, zoonotic and nonzoonotic, are widely distributed in Polish roe deer population; (2) the roe deer is the host for zoonotic Babesia (Bab. venatorum, Bab. divergens), closely related or identical with strains/species found in humans; (3) our Bab. capreoli and Bab. divergens isolates differed from reported genotypes at 2 conserved base positions, i.e., positions 631 and 663; and (4) this is the first description of Bart. schoenbuchensis infections in roe deer in Poland. We present 1 of the first complex epidemiological studies on the prevalence of Babesia, Bartonella, and A. phagocytophilum in naturally infected populations of roe deer. These game animals clearly have an important role as reservoir hosts of tick-borne pathogens, but the pathogenicity and zoonotic potential of the parasite genotypes hosted by roe deer requires further detailed investigation.
PMCID: PMC3636586  PMID: 23473225
Roe deer; Babesia; Anaplasma; Bartonella; Genetic diversity; Co-infection
12.  Reductions in Human Lyme Disease Risk Due to the Effects of Oral Vaccination on Tick-to-Mouse and Mouse-to-Tick Transmission 
Vaccinating wildlife is becoming an increasingly popular method to reduce human disease risks from pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. To successfully limit human disease risk, vaccines targeting the wildlife reservoirs of B. burgdorferi must be easily distributable and must effectively reduce pathogen transmission from infected animals, given that many animals in nature will be infected prior to vaccination. We assessed the efficacy of an easily distributable oral bait vaccine based on the immunogenic outer surface protein A (OspA) to protect uninfected mice from infection and to reduce transmission from previously infected white-footed mice, an important reservoir host of B. burgdorferi. Oral vaccination of white-footed mice effectively reduces transmission of B. burgdorferi at both critical stages of the Lyme disease transmission cycle. First, oral vaccination of uninfected white-footed mice elicits an immune response that protects mice from B. burgdorferi infection. Second, oral vaccination of previously infected mice significantly reduces the transmission of B. burgdorferi to feeding ticks despite a statistically nonsignificant immune response. We used the estimates of pathogen transmission to and from vaccinated and unvaccinated mice to model the efficacy of an oral vaccination campaign targeting wild white-footed mice. Projection models suggest that the effects of the vaccine on both critical stages of the transmission cycle of B. burgdorferi act synergistically in a positive feedback loop to reduce the nymphal infection prevalence, and thus human Lyme disease risk, well below what would be expected from either effect alone. This study suggests that oral immunization of wildlife with an OspA-based vaccine can be a promising long-term strategy to reduce human Lyme disease risk.
PMCID: PMC3610442  PMID: 23428088
Borrelia burgdorferi; Lyme disease; Oral vaccine; Outer surface protein A; Peromyscus leucopus; Tick-borne disease; White-footed mice
13.  Criteria for Identifying and Evaluating Candidate Sites for Open-Field Trials of Genetically Engineered Mosquitoes 
Recent laboratory successes in the development of genetically engineered mosquitoes for controlling pathogen transmission have fostered the need for standardized procedures for advancing the technical achievements to practical tools. It is incumbent in many cases for the same scientists doing the in-laboratory discovery research to also take on the initial challenges of developing the pathway that will move the technologies to the field. One of these challenges is having a set of criteria for selecting collaborators and sites for efficacy and safety field trials that combine rigorous science with good ethical and legal practices. Specific site-selection criteria were developed in four categories—Scientific, Regulatory, Community Engagement, and Resources—in anticipation of open-field releases of a transgenic mosquito strain designed to suppress populations of the dengue vector mosquito, Aedes aegypti. The criteria are derived from previous published material, discussions, and personal experiences with the expectation of providing guidance to laboratory scientists for addressing the conceptual and operational considerations for identifying partner researchers and countries with whom to collaborate. These criteria are not intended to be prescriptive nor can they be applied to every circumstance where genetic approaches are proposed for deployment. However, we encourage those involved in the discovery phase of research to consider each criterion during project planning activities, and where appropriate, incorporate them into a “go/no-go” decision-making process for further development and testing of the technologies.
PMCID: PMC3993056  PMID: 24689963
Genetic control; Open-release; Transgenic mosquito; Field trial; Community engagement; GMO regulation
14.  First Report of Chlamydiaceae Seroprevalence in Tibetan Pigs in Tibet, China 
The seroprevalence of Chlamydiaceae infection in Tibetan pigs in Tibet, China, was examined by indirect hemagglutination assay (IHA), between April, 2010, and December, 2010. A total of 71 of 427 serum samples (16.63%, 95% confidence interval [CI] 15.31–17.95] were positive for Chlamydiaceae antibodies. Forty Chlamydiaceae seropositives from 232 samples were recorded in sera from Nyingchi (17.24%, 95% CI 15.40–19.08) and 31 positives were recorded in 195 serum samples from Mainling (15.90%, 95% CI 14.02–17.78). The investigation showed that the prevalence in female animals was 17.61% (95% CI 15.22–20.00), and in male animals it was 12.72% (95% CI 11.07–14.37). The prevalence ranged from 0% to 20.61% (95% CI 17.81–23.48) among different age groups, with a higher prevalence in growing pigs (p<0.01). The results indicated that Chlamydiaceae infection was widespread in Tibetan pigs in Tibet, China, which is of public health concern in this region of the world. To our knowledge, this is the first report of Chlamydiaceae seroprevalence in Tibetan pigs in Tibet, China.
PMCID: PMC3601688  PMID: 23428089
Chlamydiaceae; Seroprevalence; Tibetan pigs; Tibet; Indirect hemagglutination assay
15.  Prevalence of Francisella tularensis and Francisella-Like Endosymbionts in the Tick Population of Hungary and the Genetic Variability of Francisella-Like Agents 
Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.
PMCID: PMC3601718  PMID: 23421891
Francisella-like endosymbiont; Francisella tularensis; Tick; Zoonosis
16.  Microsatellite and Mini-Exon Analysis of Mexican Human DTU I Trypanosoma cruzi Strains and Their Susceptibility to Nifurtimox and Benznidazole 
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and it affects as many as 10 million people in North and South America, where it represents a major public health problem. T. cruzi is a parasite with high genetic diversity, and it has been grouped into 6 discrete typing units (DTUs), designated as T. cruzi I (TcI) to T. cruzi VI (TcVI). Mexican isolates from humans and from vector insects have been primarily found to be TcI, and these isolates are likely to be the strains that cause the clinical manifestations observed in Mexico. However, genetic characterization and drug susceptibility assays are limited in Mexican TcI strains. In this work, 24 Mexican T. cruzi strains, obtained primarily from humans, were studied with 7 locus microsatellites and mini-exon gene by PCR. Also, drug susceptibility was evaluated by growth and mobility assays. All of the human strains belonged to TcI, and they could be further grouped through microsatellite analysis into 2 subgroups (microsatellite genotypes 1 and 2), which were not related to the host clinical status or biological origin of the strain. Two strains, both from wild mammals, belonged to the TcII–TcVI groups; these strains and the CL Brener strain constituted microsatellite genotype 3. The number of alleles in each locus was lower than reported for South American strains, and a departure from the Hardy–Weinberg equilibrium was observed. The susceptibility of these strains to nifurtimox and benznidazole was heterogeneous. T. cruzi strains characterized as microsatellite genotypes 2 and 3 were significantly more susceptible to benznidazole than strains of microsatellite genotype 1. Only 1 Mexican strain resistant to both drugs was found in this study.
PMCID: PMC3601808  PMID: 23421890
Trypanosome; Chagas disease; Genetics; Nifurtimox; Benznidazole
17.  Infection of Aedes albopictus with Chikungunya Virus Rectally Administered by Enema 
Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes albopictus and Aedes aegypti mosquitoes in tropical areas of Africa, Asia, and the islands of the Indian Ocean. In 2007 and 2009, CHIKV was transmitted outside these tropical areas and caused geographically localized infections in people in Italy and France. To temporally and spatially characterize CHIKV infection of Ae. albopictus midguts, a comparison of viral distribution in mosquitoes infected per os or by enema was conducted. Ae. albopictus infected with CHIKV LR 5′ green fluorescent protein (GFP) at a titer 106.95 tissue culture infective dose50 (TCID50)/mL, were collected and analyzed for virus dissemination by visualizing GFP expression and titration up to 14 days post inoculation (dpi). Additionally, midguts were dissected from the mosquitoes and imaged by fluorescence microscopy for comparison of midgut infection patterns between orally- and enema-infected mosquitoes. When virus was delivered via enema, the anterior midgut appeared more readily infected by 3 dpi, with increased GFP presentation observed in this same location of the midgut at 7 and 14 dpi when compared to orally-infected mosquitoes. This work demonstrates that enema delivery of virus is a viable technique for use of mosquito infection. Enema injection of mosquitoes may be an alternative to intrathoracic inoculation because the enema delivery more closely models natural infection and neither compromises midgut integrity nor involves a wound that can induce immune responses. Furthermore, unlike intrathoracic delivery, the enema does not bypass midgut barriers to infect tissues artificially in the hemocoel of the mosquito.
PMCID: PMC3564464  PMID: 23249139
Chikungunya virus; Aedes albopictus; Aedes aegypti; Enema injection
18.  Human Puumala and Dobrava Hantavirus Infections in the Black Sea Region of Turkey: A Cross-Sectional Study 
This study was carried out to better understand the epidemiology of hantaviruses in a province of Turkey (Giresun) where human hantavirus disease has recently been detected. In this cross-sectional study, a total of 626 blood samples from healthy people aged 15 and 84 years old were collected both in urban and rural areas in 2009. The sera were tested by enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and the focus reduction neutralization test (FRNT). We screened the samples by an ELISA and found that 65/626 samples reacted positively for the presence of hantavirus-reactive immunoglobulin G (IgG). Twenty of the 65 ELISA-positive samples could be confirmed by an immunobloting assay, and the overall seroprevalence was thereby calculated to 3.2% (20/626). The seroprevalence of the people living in wood areas or adobe houses 9/17 (52.9%) was significantly higher than among people living in concrete houses 10/47 (21.3%) (p=0.014). Finally, 3 of the 20 immunoblot-positive sera were confirmed as specific for the Puumala hantavirus serotype by FRNT, 1 serum was confirmed as Dobrava virus-specific, whereas 1 serum was found to be equally reactive to Dobrava and Saaremaa viruses. We will now focus on further investigations of the ecology and epidemiology of hantaviruses in humans and their carrier animals in Turkey, studies that have already been started and will be further intensified.
PMCID: PMC3564479  PMID: 23289396
Hantavirus; Serology; Turkey
19.  Pre-Exposure Rabies Vaccination among US International Travelers: Findings from the Global TravEpiNet Consortium 
Background: People who travel to areas with high rabies endemicity and have animal contact are at increased risk for rabies exposure. We examined characteristics of international travelers queried regarding rabies vaccination during pretravel consultations at Global TravEpiNet (GTEN) practices during 2009–2010.
Material and Methods: We performed bivariate and multivariable analyses of data collected from 18 GTEN clinics. Travel destinations were classified by strength level of rabies vaccination recommendation.
Results: Of 13,235 travelers, 226 (2%) reported previous rabies vaccination, and 406 (3%) received rabies vaccine at the consultation. Common travel purposes for these 406 travelers were leisure (26%), research/education (17%), and nonmedical service work (14%). Excluding the 226 who were previously vaccinated, 8070 (62%) of 13,009 travelers intended to visit one or more countries with a strong recommendation for rabies vaccination; 1675 (21%) of these 8070 intended to travel for 1 month or more. Among these 1675 travelers, 145 (9%) were vaccinated, 498 (30%) declined vaccination, 832 (50%) had itineraries that clinicians determined did not indicate vaccination, and 200 (12%) remained unvaccinated for other reasons. In both bivariate and multivariate analyses, travelers with trip durations >6 months versus 1–3 months (adjusted odds ratio [OR]=4.9 [95% confidence interval [CI] 2.1, 11.4]) and those traveling for “research/education” or to “provide medical care” (adjusted OR=5.1 [95% CI 1.9, 13.7] and 9.5 [95% CI 2.2, 40.8], respectively), compared with leisure travelers, were more likely to receive rabies vaccination.
Conclusions: Few travelers at GTEN clinics received rabies vaccine, although many planned trips 1 month long or more to a strong-recommendation country. Clinicians often determined that vaccine was not indicated, and travelers often declined vaccine when it was offered. The decision to vaccinate should take into account the strength of the vaccine recommendation at the destination country, duration of stay, availability of postexposure prophylaxis, potential for exposure to animals, and likelihood of recurrent travel to high-risk destinations.
PMCID: PMC3928762  PMID: 24359420
Rabies vaccine; Travelers; Travel clinics; International destinations
20.  Detection of Rickettsia massiliae in Rhipicephalus sanguineus from the Eastern United States 
We report the first evidence of Rickettsia massiliae in the brown dog tick, Rhipicephalus sanguineus, from the East Coast of the United States. As part of routine pathogen surveillance, DNA samples from ixodid ticks were tested for spotted fever group rickettsiae by nested PCR. A R. massiliae-positive tick was collected off a beagle mix recently rescued from North Carolina. Infection was confirmed by partial sequence analysis of the htrA, gltA, ompB, ompA, and sca4 genes, which had 100% identity to a R. massiliae isolate from Arizona.
PMCID: PMC3540894  PMID: 23199270
Rickettsia massiliae; Rhipicephalus sanguineus
21.  Ecology of Catarina Virus (family Arenaviridae) in Southern Texas, 2001–2004 
A total of 3941 rodents were captured during a 46-month prospective (mark-recapture) study on the ecology of Catarina virus in southern Texas. Antibody reactive against Catarina virus was found in 73 (11.9%) of 611 southern plains woodrats (Neotoma micropus) and none of 3330 other rodents; strains of Catarina virus were isolated from 6 antibody-negative and 9 antibody-positive southern plains woodrats; and the infections in at least 3 southern plains woodrats were chronic. These results affirm the notion that the southern plains woodrat is the principal host of Catarina virus and suggest that Catarina virus infection is highly specific to N. micropus.
PMCID: PMC3540900  PMID: 23210637
Arenavirus; Arenaviridae; Catarina virus; Neotoma micropus; Southern plains woodrat; Tacaribe serocomplex
22.  Japanese Encephalitis Virus in Mosquitoes and Swine in Yunnan Province, China 2009–2010 
The residential regions of Yunnan province, canton of Jing Hong, in China were surveyed for Japanese encephalitis virus (JEV) infection in mosquito and swine vectors to determine the frequency of JEV-carrying zoonotic vectors in 2009–2010. A total of 21,500 mosquitoes were collected and divided by species, and brain tissue was collected from 108 stillborn piglets. The infection rates for the different JEV species were 13.2% for Culex tritaeniorhynchus, 2.7% for Anopheles sinensis, 0.7% for Armigeres subalbatus, and 18.5% for stillborn piglets. The complete genomes of two JEV samples that were collected in different seasons and different regions, Yunnan 0901 and Yunnan 0902, were sequenced from a pool of Culex mosquitoes and stillborn piglets that had been collected randomly from several piggeries. Multiple sequence alignment with 24 fully-sequenced genes and 93 complete sequences of the JEV-encoded E gene revealed nucleotide homologies ranging from 97.2–99.6% and 94.5–99.7% in mosquitoes and piglets, respectively, and deduced amino acid homologies ranging from 97.4–98.1% and 96.0–98.2%, respectively. Phylogenetic analyses of the Yunnan 0901 and Yunnan 0902 strains' full-length genomes and E gene sequences indicated that these strains are most closely related to six Chinese SA14-derived viruses, and distantly related to the Australian FU, vellore P20778, and Japanese Ishikawa strains, and the previously isolated YN86-B8639 strains. The phylogenetic relationships based on the full-length genome were similar to those found for the E gene, indicating that phylogenetic analysis of the E gene will be a useful approach for genotyping of JEV, but not to better understand the potential changes in the biological characteristics and genetic relationship of JEV isolates.
PMCID: PMC3540924  PMID: 23199264
Genotype; Japanese encephalitis virus; Molecular epidemiology; Mosquito vector; RT-PCR
23.  Southern Plains Woodrats (Neotoma micropus) from Southern Texas Are Important Reservoirs of Two Genotypes of Trypanosoma cruzi and Host of a Putative Novel Trypanosoma Species 
Trypanosoma cruzi, the causative agent of Chagas' disease, is an important public health and veterinary pathogen. Although human cases are rare in the United States, infections in wildlife, and in some areas domestic dogs, are common. In 2008 and 2010, we investigated T. cruzi prevalence in possible vertebrate reservoirs in southern Texas, with an emphasis on southern plains woodrats (Neotoma micropus). Infection status was determined using a combination of culture isolation, polymerase chain reaction (PCR), and serologic testing. Based on PCR and/or culture, T. cruzi was detected in 35 of 104 (34%) woodrats, 3 of 4 (75%) striped skunks (Mephitis mephitis), 12 of 20 (60%) raccoons (Procyon lotor), and 5 of 28 (18%) other rodents including a hispid cotton rat (Sigmodon hispidus), rock squirrel (Otospermophilus variegatus), black rat (Rattus rattus), and two house mice (Mus musculus). Additionally, another Trypanosoma species was detected in 41 woodrats, of which 27 were co-infected with T. cruzi. Genetic characterization of T. cruzi revealed that raccoon, rock squirrel, and cotton rat isolates were genotype TcIV, while woodrats and skunks were infected with TcI and TcIV. Based on the Chagas Stat-Pak assay, antibodies were detected in 27 woodrats (26%), 13 raccoons (65%), 4 skunks (100%), and 5 other rodents (18%) (two white-ankled mice [Peromyscus pectoralis laceianus], two house mice, and a rock squirrel). Seroprevalence based on indirect immunofluorescence antibody testing was higher for both woodrats (37%) and raccoons (90%), compared with the Chagas Stat-Pak. This is the first report of T. cruzi in a hispid cotton rat, black rat, rock squirrel, and white-ankled mouse. These data indicate that based on culture and PCR testing, the prevalence of T. cruzi in woodrats is comparable with other common reservoirs (i.e., raccoons and opossums) in the United States. However, unlike raccoons and opossums, which tend to be infected with a particular genotype, southern plains woodrats were infected with TcI and TcIV at near equal frequencies.
PMCID: PMC3540927  PMID: 23127189
Rodents; Triatomes; Trypanosomes; Zoonosis; Zoonotic
24.  Evidence of Antibodies to Spotted Fever Group Rickettsiae in Small Mammals and Quail from Mississippi 
Rickettsia parkeri
is a recently recognized human pathogen primarily associated with the Gulf Coast tick Amblyomma maculatum, with immature stages of this tick reported from wild vertebrates. To better understand the role of vertebrates in the natural history of this bacterium, we evaluated small mammals and ground-dwelling birds for evidence of infection with R. parkeri or exposure to the organism. We sampled small mammals (n=39) and passerines (n=47) in both north-central and southeast Mississippi, while northern bobwhite (Colinus virginianus) samples (n=31) were obtained from farms in central Mississippi. Blood from all sampled animals was tested using polymerase chain reaction (PCR) for spotted fever group rickettsiae (SFGR), and for antibodies to SFGR using R. parkeri antigen. Ectoparasite samples were removed from animals and included mites, lice, fleas, and immature ticks. Of 39 small mammal samples collected, 7 were positive for antibodies to SFGR; none tested positive by PCR for DNA of SFGR. Of 47 passerine blood samples collected, none were positive for DNA of SFGR by PCR, nor did any show serological evidence of exposure. Finally, none of 31 northern bobwhite samples tested were positive for SFGR DNA, while 7 were seropositive for rickettsial antibodies. Detection of seropositive rodents and quail suggests a role for these host species in the natural history of SFGR, possibly including R. parkeri, but the extent of their role has not yet been elucidated.
PMCID: PMC3960845  PMID: 23199266
Birds; Ectoparasites; Gulf Coast tick; Rickettsiae; Small mammals
25.  Canine Dirofilaria Infections in Two Uninvestigated Areas of Serbia: Epidemiological and Genetic Aspects 
Vector Borne and Zoonotic Diseases  2012;12(12):1031-1035.
In 2009 canine filarial infections were investigated in two northern areas of Serbia (Pančevo and Veliko Gradište), applying morphometry, biochemical staining, and immunological kit to detect Dirofilaria immitis antigens, and two home-made ELISAs to detect antibodies to D. repens and D. immitis somatic/metabolic polyproteins. Moreover, molecular tools were applied to analyze the phylogenetic relationships of the isolates. The microfilariae detected in 21/122 dogs (17.2%) were identified as D. repens (n=21) and D. immitis (n=2). D. immitis antigens were found in another 13 animals with occult infection. All of the 15 heartworm-positive dogs also had antibodies to this parasite, which were detected in another 13 subjects, indicating an overall D. immitis seroprevalence rate of 22.9%. Serology for D. repens revealed evidence of antibodies in 42.6% of the dogs, but was negative for 4 microfilaremic dogs. As for the two different areas, the prevalence of microfilariae and/or D. immitis antigens, mainly due to D. repens microfilaremic animals, was not significantly higher in Veliko Gradište (33.3%) than in Pančevo (22%). However, serology showed a different epidemiological picture. Heartworm infection occurred more often in both areas, and antibodies to dirofilarial nematodes were detected in 72.9% of dogs living in Pančevo, a rate higher than in those living in Veliko Gradište (57.1%). No risk factors for infection were found, confirming the uselessness of prophylactic drugs against D. repens, and suggesting the presence in these areas of sunrise- or sunset-biting mosquitoes as important vectors. The results indicate the need for both appropriate entomological studies and further research on the intra-species variability shown by D. repens.
PMCID: PMC3525891  PMID: 23127188
Dirofilaria; Epidemiology; Genetics; Serbia

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